1. Field of the Invention
The present invention relates to specimen holders for hydrous specimens, and to methods of using them, and in particular to specimen holders suitable for high-pressure freezing.
2. Description of Related Art
A method of rapidly freezing hydrous specimens under high pressure is generally known, for example, from German Patent DE-B 1 806 741. The advantage of freezing under high pressure as opposed to normal pressure can be explained as follows: if a pressure of about 2000 bar is applied to a specimen during cooling, the cooling rate required for vitrification (no ice crystal formation, no segregations) is reduced by a factor of one hundred, consequently making it possible to vitrify specimens in the form of slices up to a maximum thickness of 200 xcexcm. It should also be noted that, if there is any ice formation under pressure, the mesh size of the segregation patterns becomes greatly reduced, i.e. hydrous specimens about 200 xcexcm thick frozen under 2000 bar are optimally preserved ultrastructurally (nanometer range) (STUDER D., MICHEL M., WOHLWEND M., HUNZIKER E. B. and BUSCHMANN M. D., Vitrification of articular cartilage by high-pressure freezing , J. of Microscopy 179 (1295), 312-332). Thus far, the advantage of high-pressure freezing for relatively thick specimens (specimen thickness in the range of around 2 mm) has not been recognized. The reduction in the mesh size of the segregation patterns has the effect that such specimens appear optimally preserved under a light microscope, since segregations in the xcexcm range are not visible.
The specimen holder described in DE-B 1 806 741 is generally poorly suited for further processing of frozen specimens. It is a tubular, thin-walled container made, for example, of copper which is closed at one end and widened at its upper end in the form of a funnel. The specimen inside this specimen holder is subjected to pressure by a hydraulic system using a pressure transfer fluid, for example, water, and is cooled from outside by spraying on a coolant. The use of this device makes it virtually impossible for the specimens to be further processed after freezing. By applying a predetermined breaking point, it has been possible at least to use a so-called freeze etching technique. With this technique, thin metal impressions are prepared, which can be investigated.
Commercially available high-pressure freezing devices according to the prior art typically operate as follows: they use liquid nitrogen of about xe2x88x92150xc2x0 C. both as a pressure transfer medium and as a coolant. Its temperature under normal pressure is xe2x88x92196xc2x0 C.; under 2000 atm, it is solid at this temperature. The apparatus-related disadvantages of such systems, in which liquid nitrogen is used both as a pressure transfer medium and as a coolant, include the following: the machines are relatively large (about 0.8xc3x971.6xc3x971.5 m3) and heavy ( greater than 600 kg). Their use entails a risk of accidents for the operating personnel: the spraying on of over 100 ml of liquid nitrogen under 2000 bar requires relatively large bores in the pressure chamber, which accordingly has to be of a very solid construction. Accidents are known; thus far, no instances of personal injury have occurred, but property damage can run into very high FIGS. (5-20,000 Swiss Francs). The costs of these apparatuses continue to be relatively high, since they have to be produced in small numbers from high-grade materials (150-300,000Swiss Francs).
In such systems, the biological specimens are generally located in two thin-walled metal half-shells (so-called aluminum sandwich: about 3 mm outside diameter, inside diameter about 2 mm, with a variable cavity thickness of 100-600 xcexcm), which are securely clamped between two steel plates. These plates are securely bolted to a solid steel impeller (specimen holder). This specimen holder is introduced with the specimen into a high pressure chamber and arrested by a solid transverse bolt. The high pressure chamber is sealed by an O-ring on the specimen holder.
The freezing cycle in the above-described systems typically proceeds as follows: to coordinate the pressure increase and cooling, the high pressure chamber is initially filled for about 30 ms with ethanol, in order to permit the correct correlation of the pressure increase and the cooling. Then, about 100-160 ml of cold liquid nitrogen is passed by means of a high pressure cylinder through the pressure chamber in 300-600 ms. The pressure chamber has an exhaust, the diameter of which is made much smaller than the feedline. The pressure is built up by accumulation at this exhaust. If the pressure chamber were not previously filled with ethanol in the way described, the specimen would freeze before it were subjected to pressure. Ethanol in the pressure chamber is necessary for the correct correlation of the pressure build-up and cooling. It is disadvantageous in this respect that it possible for the ethanol to diffuse into the specimen and form artefacts in it or damage it. What is more, when the above-described method is employed, suspensions are often blown out of the metal half-shells.
It is often possible to achieve reproducible results by the half-shells, provided with the biological specimen, being immersed in 1-hexadecene. 1-hexadecene typically has the following advantages over aqueous solutions: no ice crystals can generally form outside the specimen; the low surface tension avoids gas bubbles, which would collapse during the pressure build-up, between the half-shells. Since 1-hexadecene is not water-miscible, the aqueous specimen is not changed during preparation. The freezing point is 4xc2x0 C., but increases under pressure (2000 atm) to about 25xc2x0 C., whereby a solid, but not rigid xe2x80x9cshellxe2x80x9d forms around the aqueous specimen. This xe2x80x9cshellxe2x80x9d is important in order not to lose the specimens during the cooling process under high pressure, since the nitrogen flow has a velocity of over 40 m/s (See, for example, STUDER D., MICHEL M. and Mxc3x9cLLER M. High-pressure freezing comes of age, Scanning Microscopy Supplement 3, Vol. 189, pages 253-269).
However, biological specimens in the form of suspensions are still often difficult to handle with the technique using 1-hexadecene. Therefore, for receiving the biological specimens, preferably cellulose capillaries of about 200 xcexcm inside diameter and 10 to 15 xcexcm wall thickness of the porous wall have been used, cut into pieces of about 2 mm in length, placed between the metal half-shells and lightly clamped in place. These cellulose capillaries are surrounded by 1-hexadecene in the specimen holder. As already described, the specimens are frozen in a pressure chamber according to the prior art and, after freezing, may be manually removed from the thin-walled, metal half-shells, which serve as specimen holders (See, for example, H. HOHENBERG, K. MANNWEILER, M. Mxc3x9cLLER, High-pressure freezing of cell suspensions in cellulose capillary tubes, Journal of Microscopy 175 (1294), 34-43, in particular FIG. 1, page 35).
The disadvantage of such specimen holders comprising metal half-shells is that the manipulation of the frozen specimens, in particular their removal from the solid 1-hexadecene, is difficult and often leads to specimens being damaged or lost. In addition, on account of their geometry, they are typically not suitable for the use of a separate circuit for the pressure transfer fluid, which in terms of apparatus is a more simple and therefore less costly means of transferring pressure to the specimen, as is described, for example, in DE-B 1 806 741.
An object of the present invention was accordingly to design specimen holders for receiving hydrous specimens, in particular in capillaries preferably made of cellulose or other materials. It was also an object to provide specimen holders which are generally suitable for use in high-pressure freezing apparatuses with separate circuits for the pressure transfer medium and coolant and to ensure easy and reliable manipulation of the frozen specimens, in particular during their removal from the specimen holder.
In accordance with these and other objectives, there is provided a specimen holder for a hydrous specimen comprising: (a) an inner hollow cylinder of a heat conductive material, (b) an inner hollow cylinder of a material which can be cut, (c) a cylindrical interior space within the inner hollow cylinder for receiving the specimen, and (d) the space between the inner hollow cylinder and an inside wall of the outer hollow cylinder being filled by a layer which is liquid at room temperature.
In yet further accordance with these and other objects, there is provided a method for freezing hydrous specimens under high pressure using a specimen holder, said method comprising: (a) introducing the hydrous specimen into a first hollow cylinder, (b) filling the cylindrical interior space of a second hollow cylinder with a substance which is liquid at room temperature, (c) manually introducing the first hollow cylinder into the interior space of the second hollow cylinder, (d) connecting the second hollow cylinder to corresponding receiving devices of a pressure transfer circuit of a high-pressure freezing system, (e) subjecting the specimen to high pressure for a short time, (f) intensely cooling the specimen for a short time so as to thereby freeze said specimen, (g) subjecting the specimen to atmospheric pressure, (h) bringing the specimen to a temperature at which the said substance melts, (i) pushing the specimen out of the second hollow cylinder by introducing an appropriately dimensioned instrument at the end of the second hollow cylinder, and (j) sending the specimen to further processing if desired.
Additional objects and advantages of the invention will be set forth in the description which follows, and in part will be obvious from the description, or may be learned by practice of the invention. The objects and advantages of the invention may be realized and obtained by means of the instrumentalities and combinations particularly pointed out in the appended claims.